Polymeric Carriers of Therapeutic Agents and Recognition Moieties for Antibody-Based Targeting of Disease Sites

ABSTRACT

The present invention concerns methods and compositions for delivery of therapeutic agents to target cells, tissues or organisms. In preferred embodiments, the therapeutic agents are delivered in the form of therapeutic-loaded polymers that may comprise many copies of one or more therapeutic agents. In more preferred embodiments, the polymer may be conjugated to a peptide moiety that contains one or more haptens, such as HSG. The agent-polymer-peptide complex may be delivered to target cells by, for example, a pre-targeting technique utilizing bispecific or multispecific antibodies or fragments, having at least one binding arm that recognizes the hapten and at least a second binding arm that binds specifically to a disease or pathogen associated antigen, such as a tumor associated antigen. Methods for synthesizing and using such therapeutic-loaded polymers and their conjugates are provided.

RELATED APPLICATIONS

This application is divisional of U.S. Patent Application Ser. No.(“USSN”) 12/766,092, filed Apr. 23, 2010, which was acontinuation-in-part of U.S. Ser. No. 11/961,436, filed Dec. 20, 2007,which claimed the benefit under 35 U.S.C. §119(e) of provisional U.S.Patent Application Ser. No. 60/885,325, filed on Jan. 17, 2007. U.S.Ser. No. 12/766,092 was a continuation-in-part of U.S. Ser. No.12/731,781 (now issued U.S. Pat. No. 8,003,111), filed Mar. 25, 2010,which claimed the benefit under 35 U.S.C. 119(e) of U.S. ProvisionalPatent Application 61/163,666, filed Mar. 26, 2009. U.S. Ser. No.12/766,092 was a continuation-in-part of U.S. Ser. No. 12/644,146 (nowissued U.S. Pat. No. 7,981,398), filed Dec. 22, 2009, which was adivisional of U.S. Ser. No. 11/925,408 (now issued U.S. Pat. No.7,666,400), filed Oct. 26, 2007. U.S. Ser. No. 12/766,092 was acontinuation-in-part of U.S. Ser. No. 12/417,917 (now issued U.S. Pat.No. 7,906,121), filed Apr. 3, 2009, which was a divisional of U.S. Ser.No. 11/478,021 (now issued U.S. Pat. No. 7,534,866), filed Jun. 29,2006. U.S. Ser. No. 12/766,092 was a continuation-in-part of U.S. Ser.No. 12/396,965 (now issued U.S. Pat. No. 7,871,622), filed Mar. 3, 2009,which was a divisional of U.S. Ser. No. 11/391,584 (now issued U.S. Pat.No. 7,521,056), filed Mar. 28, 2006. U.S. Ser. No. 12/766,092 was acontinuation-in-part of U.S. Ser. No. 12/396,605 (now issued U.S. Pat.No. 7,858,070), filed Mar. 3, 2009, which was a divisional of U.S. Ser.No. 11/633,729 (now issued U.S. Pat. No. 7,527,787), filed Dec. 5, 2006,which was a continuation-in-part of PCT/US06/010762, filed Mar. 24,2006, PCT/US06/012084, filed Mar. 29, 2006, PCT/US06/025499, filed Jun.29, 2006, U.S. Ser. No. 11/389,358 (now issued U.S. Pat. No. 7,550,143),filed Mar. 24, 2006, and claimed the benefit of U.S. Provisional PatentApplications 60/864,530, filed Nov. 6, 2006; 60/668,603, filed Apr. 6,2005; 60/728,292, filed Oct. 19, 2005; 60/751,196, filed Dec. 16, 2005;and 60/782,332, filed Mar. 14, 2006. U.S. Ser. No. 12/766,092 was acontinuation-in-part of U.S. Ser. No. 12/418,877 (now issued U.S. Pat.No. 7,906,118), filed Apr. 6, 2009, which claimed the benefit of U.S.Provisional Patent Applications 61/043,932, filed Apr. 10, 2008,61/104,916, filed Oct. 13, 2008, and 61/119,542, filed Dec. 3, 2008. Thetext of each priority application cited above is incorporated herein byreference in its entirety.

BACKGROUND

Targeting of drugs, toxins, and radionuclides to disease sites usingtumor-selective monoclonal antibodies (MAbs) is an evolving field ofbiopharmaceutical research, with three approved products impacting thepractice of medicine (Sharkey R M and Goldenberg D M, CA Cancer J. Clin.2006; 56:226-243).

Typically, a MAb for an antigen expressed on a disease site, such asthat on the surface of a tumor cell, is modified with drugs or toxins orradionuclides to form immunoconjugates, and the latter are targeted invivo. In the formation of immunoconjugates, only a limited number ofmodifying groups can be introduced on to the antibody without affectingthe MAb's immunoreactivity. Moreover, many of these modifiers, such asdrugs, are generally hydrophobic, and cause solubility problems if thesubstitution is increased beyond a threshold level. These problems havebeen addressed by loading drugs or other moieties on to a water-solublepolymer such as dextran, and subsequently covalently linking thedrug-polymer to antibodies to the Fc region carbohydratessite-specifically. See Shih, et al., U.S. Pat. No. 4,699,784 and U.S.Pat. No. 5,057,313, both incorporated herein by reference in theirentirety. The size of the directly conjugated antibody-polymer-drugconstruct can be an issue in certain applications, and an alternativeapproach to increasing the concentration of the drugs at the diseasesite, other than using a direct immunoconjugate, is desirable.

An approach that bypasses the limitations of using directimmunoconjugates, called ‘pretargeting’, makes use of a bi- ormultispecific antibody with specificities for disease antigens as wellas for a small molecular mass hapten (Goldenberg D M, et al., J ClinOncol. 2006; 24: 823-834). In this method, the disease targeting step istemporally separated from the targeting of the drug molecule. Briefly, abispecific or multispecific antibody is administered first to a patient.After the antibody localizes at the disease site by binding todisease-associated antigen, a second agent consisting of the drugattached to the small molecular mass hapten is administered. Thisdrug-attached hapten selectively binds to the anti-hapten component ofthe bispecific antibody that has been pretargeted at the disease site.Generally, the second step agent is a small molecule, such as a peptidewith hapten and drug attached to it, which clears rapidly fromcirculation, with a single or just a few passes at the tumor site wherethe material must be captured. In addition, the usual design of suchsecond step agents results in only a few drug molecules attached. Thecombination of quick clearance and low drug substitution results in lowspecific activity of the drug at the disease site.

There thus exists a need for developing new methods for targeting alarge number of therapeutic agents to disease sites selectively. Ageneral method, applicable to both direct immunoconjugate as well as thesecond step agent of pretargeting approach, would be highly desirable.

SUMMARY

The present invention solves the aforementioned problems of direct orpretargeting mode of antibody-based delivery of therapeutics byproviding a therapeutic-loaded polymer that is also covalently attachedto a low molecular weight peptide. For application to pretargeting, thepeptide moiety may contain one or two hapten units, such as HSG(histamine-succinyl-glycine). The use of bispecific antibodies fordiagnosis and therapy, illustrated with anti-HSG antibody as one arm ofthe bispecific is well known in the art, and methods for the preparationof HSG-containing peptides are also described in the art (U.S. Pat. Nos.7,138,103 and 7,172,751, both incorporated herein by reference in theirentirety).

For use with direct immunoconjugates, the peptide may contain functionalgroup(s) for covalent linking to bi- or multivalent antibodies, orfragments thereof, in a manner that does not affect the antigen-bindingproperties of antibodies. In a preferred embodiment, the peptide may beattached to bi- or multivalent antibodies or fragments thereof using the‘dock and lock (DNL)’ technology (Rossi E A, et al., Proc Natl Acad SciUSA 2006; 103:6841-6846; U.S. Patent Application Publication Nos.20060228300; 20070086942 and 20070140966, the text of each of which isincorporated herein by reference in its entirety). These and otheraspects of the invention are described in detail below.

DETAILED DESCRIPTION

In preferred embodiments, the polymer, such as a dextran molecule, isderivatized to possess multiple carboxylic acid groups. A fraction ofthese carboxylic acid groups is derivatized by amide formation withethylenediamine such that about one molecule of a maleimide-containingcross-linker is attached per molecule of the polymer. The remainingcarboxylic acid groups are modified to possess a pre-determined level(substitution) of a functional group that is chemoselective forattachment to a drug. The substitution level of this functional groupwill determine the substitution level of drugs attached to the polymer.

In one embodiment, the functional group on the polymer is an acetylenemoiety. The polymer-(alkyne)_(x)-peptide derivative is coupled with anazide-containing drug in a copper (+1)-catalyzed cycloaddition reactioncalled ‘click chemistry’ (Kolb H C and Sharpless K B, Drug Discov Today2003; 8: 1128-37). Click chemistry takes place in aqueous solution atnear-neutral pH conditions, and is thus amenable for drug conjugation.The advantage of click chemistry is that it is chemoselective, andcomplements other well-known conjugation chemistries such as thethiol-maleimide reaction. The attachment of drug to the polymer-peptideaddend is carried out as a final step in the preparation of material forpretargeting. In the immunoconjugate formation in the context of the DNLapproach, the drug can be attached to the polymer prior to DNL assembly.It can be also more advantageously performed as a final step after theDNL assembly, and this way the drug is not involved during the DNLprocess.

In another embodiment, the functional group on the polymer is ahydrazide. The drug such as doxorubicin, containing a keto group, can becoupled to the hydrazide-appended polymer at a pH in the range of5-to-7.

In a third embodiment, the functional group on the polymer is acyclodextrin molecule that can non-covalently bind to drugs byhost-guest complexation.

In some embodiments, the polymer can be substituted with 2 or moredrugs. This is particularly suited for the click chemistry approachwhereby a single polymer addend with multiple alkyne moieties (usuallymonosubstituted acetylenes) can be first coupled with oneazide-containing drug. By limiting the molar equivalents, only a certainfraction of the acetylene groups are derivatized by the firstdrug-azide. The process is repeated with a second azide-containing drugso that the remaining acetylene groups are coupled. For example, thefirst drug can be doxorubicin which is a topoisomerase II inhibitor, andthe second drug can be SN-38 which is a topoisomerase I inhibitor.

When attached to the polymer by the click chemistry method, the bondingis via a stable triazole. A cleavable linker may additionally be builtinto the cross-linker between the drug and the azide to enable drugrelease.

Embodiments with respect to the nature of the ‘recognition moiety’ areas follows: (1) It can be a peptide containing one or 2 molecules of ahapten such as HSG or DTPA, that binds specifically to anti-HSG oranti-DTPA antibodies, respectively. The drug-polymer-hapten can then beused in a pretargeting mode after first targeting the disease site witha bi- or multispecific antibody possessing at least one arm specific forthe disease site and at least one arm specific for the hapten.Alternatively, a pre-complexed multispecific antibody-polymer-hapten maybe utilized within the scope of this invention. (2) It can be folicacid, such that the polymer-drug-folate complex is used to target folatereceptors on disease sites such as in cancers, in as much as targetingof folate receptors using folate-appended diagnostic or therapeuticmoieties is a well known strategy. (3) It can be a peptide such assomatostatin (SS) or VIP peptide, useful for receptor-targeting atdisease sites. (4) It can be biotin, for use inavidin/streptavidin-based pretargeting protocols. (5) It can be acomplementary antisense oligonucleotide. (6) It can be the anchoringdomain (AD) peptide of the ‘dock and lock’ (DNL) methodology (see, e.g.,U.S. patent application Ser. Nos. 11/389,358, filed Mar. 24, 2006;11/391,584, filed Mar. 28, 2006; 11/478,021, filed Jun. 29, 2006; and11/633,729, filed Dec. 5, 2006, each incorporated herein by reference inits entirety). The components specific for the ‘recognition moieties’and part of the bi- or multispecific antibodies used in pretargetingprotocol using embodiments 1 through 5 described in this paragraph areanti-HSG or anti-DTPA antibody; anti-folate antibody; anti-somatostatinantibody; avidin/streptavidin; or oligonucleotide, respectively. Thecounterpart component of the sixth embodiment is defined by the natureof the DNL methodology and for the AD sequence would be a complementaryDDD sequence. In embodiments 2 and 3, the polymer-drug-folate orpolymer-drug-SS can latch on to the bi- or multispecific antibodypretargeted at the disease site and also target the folate or SSreceptors, respectively, thereby augmenting the mechanisms of targetingat the disease sites. The number of such recognition moieties introducedon to the polymer is preferably 1-10, more preferably 1-5, and mostpreferably 1-2. The number of recognition moieties per polymer ispreferably 1 when using in the context of DNL assemblage, but can begreater than 1 when used in pretargeting formats.

Examples of drug-dextran are shown below. Scheme 1 gives a generalapproach to modification of polymer using acetylene-azide couplingchemistry, and is illustrated by structures 1 through 3.

Alternatively, the polymer can be derivatized to contain an azide groupin place of acetylene, and the drug can be derivatized with acetylenegroup instead of azide.

Structure 4: This represents one type of linking by the ‘clickchemistry’ to one type of drug. In this, ‘Rm’ is a recognition moiety,n=0˜16, x=10-1000, and ‘(Z)’ is additional spacer consisting of(CH₂)_(m)—NH—CO moiety, where m is an integer with values of 1-20,preferably 1-5, and most preferably 1.

Structure-4

Structure 5: This represents one type of linking by the ‘clickchemistry’ to 2 types of drugs (the ‘recognition’ moiety indicated byam'). Drug-1 can be an anthracycline drug, such as doxorubicin, which isa topoisomerase II inhibitor, while the second drug can be acamptothecin, such as SN-38, which is a topoisomerase I inhibitor. Inthis example, ‘x’ is the repeating dextran unit defined by the polymersize, ‘n’ is the number of moieties derivatized with drug 1 and drug 2,which defines the level of drug loading, and ‘Z’ is spacer. Althoughshown in this structure as ‘n’ for both drug 1 and drug 2, the value of‘n’ can differ for drug 1 and drug 2 for different levels of the drugloadings. The acetylene-azide coupling results in a triazole structuralmoiety as shown. The spacer 1 and spacer 2 contain cleavable linkerpart. The cleavable linker can be an acid-cleavable hydrazone orcathepsin B cleavable peptide in the case of anthracycline such asdoxorubicin, and it can be an ester or carbonate bond and/or a cathepsinB cleavable peptide in the case of a camptothecin. The drugs can beother than that indicated, and the multiplicity of drug types is notlimited to 2. [In this structure, ‘Rm’ is a recognition moiety, n=0˜16,x=10-1000, and ‘(Z)’ is additional spacer consisting of (CH₂)_(m)—NH—COmoiety, where m is an integer with values of 1-20, preferably 1-5, andmost preferably 1.]

Structure-5

Structure 6: This is an example of chemoselective modification ofdextran. In this example of 70 KD MW dextran, 44 COOH groups are firstintroduced by reacting with 6-bromohexanoic acid, representing ‘11%’ ofmonomeric unit (or 44 moieties) modified. Of these, 20 available COOHgroups (‘5%’ of monomeric units) are converted to Boc-protectedhydrazide using BOC—NHNH₂ and water soluble carbodiimide, EDC. Theremaining COOH groups are partly converted to terminate in an amine,using ethylene diamine and EDC coupling, such that 8 amines aresubstituted per polymer. Conditions have been developed to substitutejust one of these amino groups with a modifier, such as pyridyldithiogroup of structure 7, for later attachment to a peptide.

Structure-6

Structure 7: This structure shows that an average of one SPDP moleculecan be substituted on to the 70 kD dextran. By first reacting with athiol-containing peptide in a diulfide-exchange reaction, an average ofone peptide can be introduced. Alternatively, the disulfide of structure7 can be reduced with dithiothreitol or TCEP, and the thiol-containingdextran can be reacted with a maleimide-containing peptide. Yet anothervariation is that the amine on dextran is derivatized with amaleimide-containing cross-linker for further reaction with athiol-containing peptide. The peptide moiety contains one or two haptenmolecules, such as HSG, or it is ‘AD’ peptide suitable for fusing with‘DDD’ component of DNL methodology. BOC-deprotection under acidicconditions then liberates hydrazide, suitable for reacting with aldehydeor keto group on a drug. Alternatively, and more preferably in the DNLapproach, the hydrazide moiety is replaced by acetylene group that canbe later coupled to azide-containing drug. An advantage in this approachis that the DNL assembly can be first performed, and the resultantassembly will contain drug signatures, which are actually the acetylene(or azide) groups. The DNL product can be reacted chemoselectively withan azide (or acetylene)-appended drug. An advantage of pre-assembly ofDNL product is that the drug can be defined subsequently. And, for eachassembly, containing a defined multivalent antibody component, one couldsubstitute different drug types by using the correspondingazide-derivatized drugs.

While the nature of ‘recognition moiety’ is defined in the DNL productas ‘AD’ peptide, it can be variable in other examples as enumerated in aprevious section.

Structure-7

Structure 8: This is a variation of structure 2, showing thesubstitution on dextran of cyclodextrin instead of acetylene. A suitabledrug, such as doxorubicin, capable of forming non-covalent complex withcyclodextrin is subsequently added. Cyclodextrin substitution determinesdrug substitution. [In this structure, ‘Rm’ is a recognition moiety,n=0˜16, x=10-1000, and ‘(Z)’ is additional spacer consisting of(CH₂)_(m)—NH—CO moiety, where m is an integer with values of 1-20,preferably 1-5, and most preferably 1.]

Structure-8

Structure 9: This is a variation of structure 5, showing thesubstitution on dextran of one drug via ‘click chemistry’ and thesubstitution of cyclodextrin for complexation with a second drug. As inother illustrations, ‘Rm’ is a recognition moiety, n=0˜16, x=10-1000,and ‘(Z)’ is additional spacer consisting of (CH₂)_(m)—NH—CO moiety,where m is an integer with values of 1-20, preferably 1-5, and mostpreferably 1.

Structure-9

Polymers

Water-soluble polymers such as dextran, polyglutamic acid, dendrimers,and so on, are within the scope of the invention. Although exemplifiedwith dextran, the polymer component is not limited to dextran.Polyglutamic acid already has carboxylic acid groups in it, and so it isequivalent to the carboxylic acid-added dextran from the viewpoint ofthis disclosure. Whatever strategies are described for COOH-addeddextran are equally applicable for polyglutamic acid. With differentgeneration dendrimers, functional groups are derivatized sequentially tocontain drug signatures such as alkyne or azide derivatizable withazide-drug or alkyne-drug, respectively, and other derivatives that canbe coupled to bifunctional drug derivatives.

Therapeutic Agents

Therapeutic agents for use in this invention include, for example,chemotherapeutic drugs such as vinca alkaloids, anthracyclines,epidophyllotoxins, taxanes, antimetabolites, alkylating agents,antibiotics, Cox-2 inhibitors, antimitotics, antiangiogenic andproapoptotic agents, particularly doxorubicin, methotrexate, taxol,camptothecins, and others from these and other classes of anticanceragents, and the like. Other cancer chemotherapeutic drugs includenitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acidanalogs, pyrimidine analogs, purine analogs, platinum coordinationcomplexes, hormones, and the like. Suitable chemotherapeutic agents aredescribed in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (MackPublishing Co. 1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICALBASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as wellas revised editions of these publications. Other suitablechemotherapeutic agents, such as experimental drugs, are known to thoseof skill in the art.

Exemplary drugs of use may include 5-fluorouracil, aplidin, azaribine,anastrozole, anthracyclines, bendamustine, bleomycin, bortezomib,bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin(CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin,cladribine, camptothecans, cyclophosphamide, cytarabine, dacarbazine,docetaxel, dactinomycin, daunorubicin, doxorubicin,2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin,doxorubicin glucuronide, epirubicin glucuronide, estramustine,epipodophyllotoxin, estrogen receptor binding agents, etoposide (VP16),etoposide glucuronide, etoposide phosphate, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,farnesyl-protein transferase inhibitors, gemcitabine, hydroxyurea,idarubicin, ifosfamide, L-asparaginase, lenolidamide, leucovorin,lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,nitrosurea, plicomycin, procarbazine, paclitaxel, pentostatin, PSI-341,raloxifene, semustine, streptozocin, tamoxifen, taxol, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vinorelbine,vinblastine, vincristine and vinca alkaloids.

Toxins of use may include ricin, abrin, alpha toxin, saporin,ribonuclease (RNase), e.g., onconase, DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin.

Chemokines of use may include RANTES, MCAF, MIP1-alpha, MIP1-Beta andIP-10.

In certain embodiments, anti-angiogenic agents, such as angiostatin,baculostatin, canstatin, maspin, anti-VEGF antibodies, anti-PlGFpeptides and antibodies, anti-vascular growth factor antibodies,anti-Flk-1 antibodies, anti-Flt-1 antibodies and peptides, anti-Krasantibodies, anti-cMET antibodies, anti-MIF (macrophagemigration-inhibitory factor) antibodies, laminin peptides, fibronectinpeptides, plasminogen activator inhibitors, tissue metalloproteinaseinhibitors, interferons, interleukin-12, IP-10, Gro-β, thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CM101, Marimastat, pentosan polysulphate, angiopoietin-2,interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide (roquinimex), thalidomide, pentoxifylline, genistein, TNP-470,endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine,bleomycin, AGM-1470, platelet factor 4 or minocycline may be of use.

Other useful therapeutic agents may comprise oligonucleotides,especially antisense oligonucleotides that preferably are directedagainst oncogenes and oncogene products, such as bcl-2 or p53. Apreferred form of therapeutic oligonucleotide is siRNA.

Targeting Moieties

In one embodiment, the targeting moiety may be a multivalent and/ormultispecific MAb. In another embodiment, the targeting moiety ismultivalent antibody fragment made with DNL (dock-and-lock) methodology.The targeting moiety may be a murine, chimeric, humanized, or humanmonoclonal antibody, and said antibody is in intact, fragment (Fab,Fab′, F(ab)₂, F(ab′)₂), or sub-fragment (single-chain constructs) form.

In a preferred embodiment, the targeting moiety is reactive with anantigen or epitope of an antigen expressed on a cancer or malignantcell. The cancer cell is preferably a cell from a hematopoietic tumor,carcinoma, sarcoma, melanoma or a glial tumor.

The targeting moiety is preferably an antibody (including fully human,non-human, humanized, or chimeric antibodies) or an antibody fragment(including enzymatically or recombinantly produced fragments) andbinding proteins incorporating sequences from antibodies or antibodyfragments. The antibodies, fragments, and binding proteins may bemultivalent and multispecific or multivalent and monospecific as definedabove.

In a preferred embodiment, antibodies, such as MAbs, are used thatrecognize or bind to markers or tumor-associated antigens that areexpressed at high levels on target cells and that are expressedpredominantly or only on diseased cells versus normal tissues, andantibodies that internalize rapidly. Antibodies useful within the scopeof the present invention include MAbs with properties as described above(and show distinguishing properties of different levels ofinternalization into cells and microorganisms), and contemplate the useof, but are not limited to, in cancer, the following MAbs: LL1(anti-CD74), LL2 and RFB4 (anti-CD22), RS7 (anti-epithelialglycoprotein-1 (EGP-1)), PAM-4 and KC4 (both anti-MUC1), MN-14(anti-carcinoembryonic antigen (CEA, also known as CD66e), Mu-9(anti-colon-specific antigen-p), Immu 31 (an anti-alpha-fetoprotein),TAG-72 (e.g., CC49), Tn, J591 (anti-PSMA (prostate-specific membraneantigen)), G250 (an anti-carbonic anhydrase IX MAb) and L243(anti-HLA-DR). Other useful antigens that may be targeted using theseconjugates include HER-2/neu, BrE3, CD19, CD20 (e.g., C2B8, hA20, 1F5MAbs) CD21, CD23, CD37, CD45, CD74, CD80, alpha-fetoprotein (AFP), VEGFR(e.g. Avastin®, fibronectin splice variant), ED-B (e.g., L19), EGFreceptor or ErbB1 (e.g., Erbitux®), ErbB2, ErbB3, placental growthfactor (PlGF), MUC1, MUC2, MUC3, MUC4, PSMA, gangliosides, HCG, EGP-2(e.g., 17-1A), CD37, HLA-DR, CD30, Ia, A3, A33, Ep-CAM, KS-1, Le(y),S100, PSA (prostate-specific antigen), tenascin, folate receptor,Thomas-Friedenreich antigens, tumor necrosis antigens, tumorangiogenesis antigens, Ga 733, IL-2, IL-6, T101, MAGE, insulin-likegrowth factor (ILGF), migration inhibition factor (MIF), the HLA-DRantigen to which L243 binds, CD66 antigens, i.e. CD66a-d or acombination thereof. The CD66 antigens consist of five differentglycoproteins with similar structures, CD66a-e, encoded by thecarcinoembryonic antigen (CEA) gene family members, BCG, CGM6, NCA, CGM1and CEA, respectively. These CD66 antigens are expressed mainly ingranulocytes, normal epithelial cells of the digestive tract and tumorcells of various tissues. A number of the aforementioned antigens aredisclosed in U.S. Provisional Application Ser. No. 60/426,379, entitled“Use of Multi-specific, Non-covalent Complexes for Targeted Delivery ofTherapeutics,” filed Nov. 15, 2002, incorporated herein by reference.

Exemplary anti-cancer antibodies that may be utilized include, but arenot limited to, hR1 (anti-IGF-1R, U.S. Provisional Patent ApplicationSer. No. 61/145,896, filed Jan. 20, 2009) hPAM4 (anti-MUC1, U.S. Pat.No. 7,282,567), hA20 (anti-CD20, U.S. Pat. No. 7,251,164), hA19(anti-CD19, U.S. Pat. No. 7,109,304), hIMMU31 (anti-AFP, U.S. Pat. No.7,300,655), hLL1 (anti-CD74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD22,U.S. Pat. No. 7,074,403), hMu-9 (anti-CSAp, U.S. Pat. No. 7,387,773),hL243 (anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14 (anti-CEA, U.S.Pat. No. 6,676,924), hMN-15 (anti-CEA, U.S. patent application Ser. No.10/672,278), hRS7 (anti-EGP-1, U.S. Pat. No. 7,238,785) and hMN-3(anti-CEA, U.S. Pat. No. 7,541,440) the Examples section of each citedpatent or application incorporated herein by reference. The skilledartisan will realize that this list is not limiting and any other knownantibody may be utilized in the claimed methods and compositions.

Antibodies of use may be commercially obtained from a wide variety ofknown sources. For example, a variety of antibody secreting hybridomalines are available from the American Type Culture Collection (ATCC,Manassas, Va.). A large number of antibodies against various diseasetargets, including but not limited to tumor-associated antigens, havebeen deposited at the ATCC and/or have published variable regionsequences and are available for use in the claimed methods andcompositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803;7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018;6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244;6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533;6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580;6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226;6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206;6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852;6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618;6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227;6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408;6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;6,455,044; 6,455,040, 6,451,310; 6,444,206′ 6,441,143; 6,432,404;6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091;6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654;6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393;6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289;6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554;5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953,5,525,338. These are exemplary only and a wide variety of otherantibodies and their hybridomas are known in the art. The skilledartisan will realize that antibody sequences or antibody-secretinghybridomas against almost any disease-associated antigen may be obtainedby a simple search of the ATCC, NCBI and/or USPTO databases forantibodies against a selected disease-associated target of interest. Theantigen binding domains of the cloned antibodies may be amplified,excised, ligated into an expression vector, transfected into an adaptedhost cell and used for protein production, using standard techniqueswell known in the art.

In another preferred embodiment of the present invention involvingpolymer-therapeutic-recognition moiety precomplexed or fused by the DNLmethodology, antibodies are used that internalize rapidly and are thenre-expressed, processed and presented on cell surfaces, enablingcontinual uptake and accretion of circulating conjugate by the cell. Anexample of a most-preferred antibody/antigen pair is LL1, an anti-CD74MAb (invariant chain, class II-specific chaperone, Ii). The CD74 antigenis highly expressed on B-cell lymphomas, certain T-cell lymphomas,melanomas and certain other cancers (Ong et al., Immunology 98:296-302(1999)), as well as certain autoimmune diseases. This embodiment isparticularly preferred as a pre-complexed or DNL construct incorporatingpolymer-therapeutic-recognition moiety.

In various embodiments, a conjugate as disclosed herein may be part of acomposite, multispecific antibody. Such antibodies may contain two ormore different antigen binding sites, with differing specificities. Themultispecific composite may bind to different epitopes of the sameantigen, or alternatively may bind to two different antigens. Some ofthe more preferred target combinations include the following. This is alist of examples of preferred combinations, but is not intended to beexhaustive.

TABLE 1 Some Examples of multispecific antibodies First target Secondtarget MIF A second proinflammatory effector cytokine, especiallyHMGB-1, TNF-α, IL-1, or IL-6 MIF Proinflammatory effector chemokine,especially MCP-1, RANTES, MIP-1A, or MIP-1B MIF Proinflammatory effectorreceptor, especially IL-6R IL-13R, and IL-15R MIF Coagulation factor,especially TF or thrombin MIF Complement factor, especially C3, C5, C3a,or C5a MIF Complement regulatory protein, especially CD46, CD55, CD59,and mCRP MIF Cancer associated antigen or receptor HMGB-1 A secondproinflammatory effector cytokine, especially MIF, TNF-α, IL-1, or IL-6HMGB-1 Proinflammatory effector chemokine, especially MCP-1, RANTES,MIP-1A, or MIP-1B HMGB-1 Proinflammatory effector receptor especiallyMCP-1, RANTES, MIP-1A, or MIP-1B HMGB-1 Coagulation factor, especiallyTF or thrombin HMGB-1 Complement factor, especially C3, C5, C3a, or C5aHMGB-1 Complement regulatory protein, especially CD46, CD55, CD59, andmCRP HMGB-1 Cancer associated antigen or receptor TNF-α A secondproinflammatory effector cytokine, especially MIF, HMGB-1, TNF-α, IL-1,or IL-6 TNF-α Proinflammatory effector chemokine, especially MCP-1,RANTES, MIP-1A, or MIP-1B TNF-α Proinflammatory effector receptor,especially IL-6R IL-13R, and IL-15R TNF-α Coagulation factor, especiallyTF or thrombin TNF-α Complement factor, especially C3, C5, C3a, or C5aTNF-α Complement regulatory protein, especially CD46, CD55, CD59, andmCRP TNF-α Cancer associated antigen or receptor LPS Proinflammatoryeffector cytokine, especially MIF, HMGB-1, TNF-α, IL-1, or IL-6 LPSProinflammatory effector chemokine, especially MCP-1, RANTES, MIP-1A, orMIP-1B LPS Proinflammatory effector receptor, especially IL-6R IL-13R,and IL-15R LPS Coagulation factor, especially TF or thrombin LPSComplement factor, especially C3, C5, C3a, or C5a LPS Complementregulatory protein, especially CD46, CD55, CD59, and mCRP TF or thrombinProinflammatory effector cytokine, especially MIF, HMGB-1, TNF-α, IL-1,or IL-6 TF or thrombin Proinflammatory effector chemokine, especiallyMCP-1, RANTES, MIP-1A, or MIP-1B TF or thrombin Proinflammatory effectorreceptor, especially IL-6R IL-13R, and IL-15R TF or thrombin Complementfactor, especially C3, C5, C3a, or C5a TF or thrombin Complementregulatory protein, especially CD46, CD55, CD59, and mCRP TF or thrombinCancer associated antigen or receptor

Still other combinations, such as are preferred for cancer therapies,include CD20+CD22 antibodies, CD74+CD20 antibodies, CEACAM5(CEA)+CEACAM6 antibodies, insulin-like growth factor (ILGF)+CEACAM5antibodies, EGP-1 (e.g., RS-7)+ILGF antibodies, CEACAM5+EGFR antibodies.Such antibodies need not only be used in combination, but can becombined as fusion proteins of various forms, such as IgG, Fab, scFv,and the like, as described in U.S. Pat. Nos. 6,083,477; 6,183,744 and6,962,702 and U.S. Patent Application Publication Nos. 20030124058;20030219433; 20040001825; 20040202666; 20040219156; 20040219203;20040235065; 20050002945; 20050014207; 20050025709; 20050079184;20050169926; 20050175582; 20050249738; 20060014245 and 20060034759, eachof which is incorporated herein by reference in their entirety.

In certain embodiments, the binding moieties described herein maycomprise one or more avimer sequences. Avimers are a class of bindingproteins somewhat similar to antibodies in their affinities andspecificities for various target molecules. They were developed fromhuman extracellular receptor domains by in vitro exon shuffling andphage display. (Silverman et al., 2005, Nat. Biotechnol. 23:1493-94;Silverman et al., 2006, Nat. Biotechnol. 24:220.) The resultingmultidomain proteins may comprise multiple independent binding domains,which may exhibit improved affinity (in some cases sub-nanomolar) andspecificity compared with single-epitope binding proteins. (Id.) Invarious embodiments, avimers may be attached to, for example, AD and/orDDD sequences for use in the claimed methods and compositions, asdescribed in provisional U.S. Patent Application Ser. Nos. 60/668,603,filed Apr. 6, 2005 and 60/751,196, filed Dec. 16, 2005, eachincorporated herein in their entirety by reference. Additional detailsconcerning methods of construction and use of avimers are disclosed, forexample, in U.S. Patent Application Publication Nos. 20040175756,20050048512, 20050053973, 20050089932 and 20050221384, the Examplessection of each of which is incorporated herein by reference.

In a preferred embodiment, an intracellularly-cleavable moietyincorporated in the ‘drug-polymer-recognition moiety’ may be cleavedafter its conjugate with the pretargeted multispecific antibody, or itsnon-covalent complex with the multispecific antibody, or a covalent DNLconstruct is internalized into the cell, and particularly cleaved byesterases and peptidases or by pH-dependent processes or by disulfidereduction.

Therapeutic Methods

Another embodiment relates to a method of treating a subject, comprisingadministering a therapeutically effective amount of a therapeuticconjugate of the preferred embodiments of the present invention to asubject. Diseases that may be treated with the therapeutic conjugates ofthe preferred embodiments include, but are not limited to B-cellmalignancies (e.g., non-Hodgkin's lymphoma and chronic lymphocyticleukemia using, for example LL2 MAb; see U.S. Pat. No. 6,183,744),adenocarcinomas of endodermally-derived digestive system epithelia,cancers such as breast cancer and non-small cell lung cancer, and othercarcinomas, sarcomas, glial tumors, myeloid leukemias, etc. Inparticular, antibodies against an antigen, e.g., an oncofetal antigen,produced by or associated with a malignant solid tumor or hematopoieticneoplasm, e.g., a gastrointestinal, lung, breast, prostate, ovarian,testicular, brain or lymphatic tumor, a sarcoma or a melanoma, areadvantageously used. Such therapeutics can be given once or repeatedly,depending on the disease state and tolerability of the conjugate, andcan also be used optimally in combination with other therapeuticmodalities, such as surgery, external radiation, radioimmunotherapy,immunotherapy, chemotherapy, antisense therapy, interference RNAtherapy, gene therapy, and the like. Each combination will be adapted tothe tumor type, stage, patient condition and prior therapy, and otherfactors considered by the managing physician.

As used herein, the term “subject” refers to any animal (i.e.,vertebrates and invertebrates) including, but not limited to mammals,including humans. The term subject also includes rodents (e.g., mice,rats, and guinea pigs). It is not intended that the term be limited to aparticular age or sex. Thus, adult and newborn subjects, as well asfetuses, whether male or female, are encompassed by the term.

In various embodiments, antibodies against known tumor-associatedantigens as described above may be utilized for therapy of diseases,such as cancer. For example, the diseases that are preferably treatedwith anti-CD74 antibodies include, but are not limited to, non-Hodgkin'slymphoma, Hodgkin's disease, melanoma, lung cancer, myeloid leukemias,and multiple myeloma. Continual expression of the CD74 antigen for shortperiods of time on the surface of target cells, followed byinternalization of the antigen, and re-expression of the antigen,enables the targeting LL1 antibody to be internalized along with anychemotherapeutic moiety it carries. This allows a high, and therapeutic,concentration of LL1-chemotherapeutic drug conjugate to be accumulatedinside such cells. Internalized LL1-chemotherapeutic drug conjugates arecycled through lysosomes and endosomes, and the chemotherapeutic moietyis released in an active form within the target cells.

In another preferred embodiment, therapeutic conjugates comprising theMu-9 MAb can be used to treat colorectal, as well as pancreatic andovarian cancers as disclosed in U.S. application Ser. No. 10/116,116,filed Apr. 5, 2002 and by Gold et al. (Cancer Res. 50: 6405 (1990), andreferences cited therein). In addition, the therapeutic conjugatescomprising the PAM-4 MAb can be used to treat pancreatic cancer, asdisclosed in U.S. Provisional Application Ser. No. 60/388,314, filedJun. 14, 2002.

In another preferred embodiment, the therapeutic conjugates comprisingthe RS-7 MAb can be used to treat carcinomas such as carcinomas of thelung, stomach, urinary bladder, breast, ovary, uterus, and prostate, asdisclosed in U.S. Provisional Application Ser. No. 60/360,229, filedMar. 1, 2002 and by Stein et al. (Cancer Res. 50: 1330 (1990) andAntibody Immunoconj. Radiopharm. 4: 703 (1991)).

In another preferred embodiment, the therapeutic conjugates comprisingthe anti-AFP MAb can be used to treat hepatocellular carcinoma, germcell tumors, and other AFP-producing tumors using humanized, chimericand human antibody forms, as disclosed in U.S. Provisional ApplicationSer. No. 60/399,707, filed Aug. 1, 2002.

In another preferred embodiment, the therapeutic conjugates comprisinganti-tenascin antibodies can be used to treat hematopoietic and solidtumors and conjugates comprising antibodies to Le(y) can be used totreat solid tumors.

In a preferred embodiment, the antibodies that are used in the treatmentof human disease are human or humanized (CDR-grafted) versions ofantibodies; although murine and chimeric versions of antibodies can beused. Same species IgG molecules as delivery agents are mostly preferredto minimize immune responses. This is particularly important whenconsidering repeat treatments. For humans, a human or humanized IgGantibody is less likely to generate an anti-IgG immune response frompatients. Antibodies such as hLL1 and hLL2 rapidly internalize afterbinding to internalizing antigen on target cells, which means that thechemotherapeutic drug being carried is rapidly internalized into cellsas well. However, antibodies that have slower rates of internalizationcan also be used to effect selective therapy with this invention.

In another preferred embodiment, the therapeutic conjugates can be usedagainst pathogens, since antibodies against pathogens are known. Forexample, antibodies and antibody fragments which specifically bindmarkers produced by or associated with infectious lesions, includingviral, bacterial, fungal and parasitic infections, for example caused bypathogens such as bacteria, rickettsia, mycoplasma, protozoa, fungi, andviruses, and antigens and products associated with such microorganismshave been disclosed, inter alia, in Hansen et al., U.S. Pat. No.3,927,193 and Goldenberg U.S. Pat. Nos. 4,331,647, 4,348,376, 4,361,544,4,468,457, 4,444,744, 4,818,709 and 4,624,846, and in Reichert andDewitz, cited above. In a preferred embodiment, the pathogens areselected from the group consisting of HIV virus causing AIDS,Mycobacterium tuberculosis, Streptococcus agalactiae,methicillin-resistant Staphylococcus aureus, Legionella pneumophilia,Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhosae,Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponemapallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus,cytomegalovirus, herpes simplex virus I, herpes simplex virus II, humanserum parvo-like virus, respiratory syncytial virus, varicella-zostervirus, hepatitis B virus, measles virus, adenovirus, human T-cellleukemia viruses, Epstein-Barr virus, murine leukemia virus, mumpsvirus, vesicular stomatitis virus, sindbis virus, lymphocyticchoriomeningitis virus, wart virus, blue tongue virus, Sendai virus,feline leukemia virus, reo virus, polio virus, simian virus 40, mousemammary tumor virus, dengue virus, rubella virus, West Nile virus,Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosomarangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosomabrucei, Schistosoma mansoni, Schistosoma japanicum, Babesia bovis,Elmeria tenella, Onchocerca volvulus, Leishmania tropica, Trichinellaspiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taeniasaginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasmaarthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasmalaidlawii, M. salivarium and M. pneumoniae, as disclosed in U.S. Pat.No. 6,440,416.

In a more preferred embodiment, drug conjugates comprising anti-gp120and other such anti-HIV antibodies can be used as therapeutics for HIVin AIDS patients; and drug conjugates of antibodies to Mycobacteriumtuberculosis are suitable as therapeutics for drug-refractivetuberculosis. Fusion proteins of anti-gp120 MAb (anti HIV MAb) and atoxin, such as Pseudomonas exotoxin, have been examined for antiviralproperties (Van Oigen et al., J Drug Target, 5:75-91, 1998)). Attemptsat treating HIV infection in AIDS patients failed possibly due toinsufficient efficacy or unacceptable host toxicity. The drug conjugatesof the present invention advantageously lack such toxic side effects ofprotein toxins, and are therefore advantageously used in treating HIVinfection in AIDS patients. These drug conjugates can be given alone orin combination with other antibiotics or therapeutic agents that areeffective in such patients when given alone.

In another preferred embodiment, diseases that may be treated using thetherapeutic conjugates include, but are not limited to immunedysregulation disease and related autoimmune diseases, including ClassIII autoimmune diseases such as immune-mediated thrombocytopenias, suchas acute idiopathic thrombocytopenic purpura and chronic idiopathicthrombocytopenic purpura, dermatomyositis, Sjögren's syndrome, multiplesclerosis, Sydenham's chorea, myasthenia gravis, systemic lupuserythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosum, Takayasu'sarteritis, Addison's disease, rheumatoid arthritis, sarcoidosis,ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritisnodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangitisubiterans, Sjögren's syndrome, primary biliary cirrhosis, Hashimoto'sthyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis,rheumatoid arthritis, polymyositis/dermatomyositis, polychondritis,pamphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis and fibrosing alveolitis, and also juvenile diabetes,as disclosed in U.S. Provisional Application Ser. No. 60/360,259, filedMar. 1, 2002. Typical antibodies useful in these diseases include, butare not limited to, those reactive with HLA-DR antigens or B-cell orT-cell antigens (e.g., CD19, CD20, CD21, CD22, CD23, CD4, CD5, CD8,CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40,CD40L, CD46, CD52, CD54, CD74, CD80, CD126, B7, MUC1, Ia, HM1.24, andHLA-DR). Since many of these autoimmune diseases are affected byautoantibodies made by aberrant B-cell populations, depletion of theseB-cells by therapeutic conjugates involving such antibodies bound withthe drugs used in this invention, is a preferred method of autoimmunedisease therapy, especially when B-cell antibodies are combined, incertain circumstances, with HLA-DR antibodies and/or T-cell antibodies(including those which target IL-2 as an antigen, such as anti-TACantibody). In a preferred embodiment, the anti-B-cell, anti-T-cell, oranti-macrophage or other such antibodies of use in the treatment ofpatients with autoimmune diseases also can be conjugated to result inmore effective therapeutics to control the host responses involved insaid autoimmune diseases, and can be given alone or in combination withother therapeutic agents, such as TNF inhibitors or TNF antibodies,unconjugated B- or T-cell antibodies, and the like.

In a preferred embodiment, diseases that may be treated using thetherapeutic conjugates include cardiovascular diseases, such as fibrinclots, atherosclerosis, myocardial ischemia and infarction. Antibodiesto fibrin are known and in clinical trials as imaging agents fordisclosing said clots and pulmonary emboli, while anti-granulocyteantibodies, such as MN-3, MN-15, NCA95, and CD15 antibodies, can targetmyocardial infarcts and myocardial ischemia, while anti-macrophage,anti-low-density lipoprotein (LDL), and anti-CD74 (e.g., hLL1)antibodies can be used to target atherosclerotic plaques.

In yet another preferred embodiment, diseases that may be treated usingthe therapeutic conjugates include neurodegenerative diseasescharacterized by a specific lesions against which a targeting moiety canbe used, such as amyloid or beta-amyloid associated with Alzheimer'sdisease, and which serves as a target for localizing antibodies.

In a preferred embodiment, a more effective incorporation into cells andpathogens can be accomplished by using multivalent, multispecific ormultivalent, monospecific antibodies. Multivalent means the use ofseveral binding arms against the same or different antigen or epitopeexpressed on the cells, whereas multispecific antibodies involve the useof multiple binding arms to target at least two different antigens orepitopes contained on the targeted cell or pathogen. Examples of suchbivalent and bispecific antibodies are found in U.S. patent applications60/399,707, filed Aug. 1, 2002; 60/360,229, filed Mar. 1, 2002;60/388,314, filed Jun. 14, 2002; and 10/116,116, filed Apr. 5, 2002, allof which are incorporated by reference herein. These multivalent ormultispecific antibodies are particularly preferred in the targeting ofcancers and infectious organisms (pathogens), which express multipleantigen targets and even multiple epitopes of the same antigen target,but which often evade antibody targeting and sufficient binding forimmunotherapy because of insufficient expression or availability of asingle antigen target on the cell or pathogen. By targeting multipleantigens or epitopes, said antibodies show a higher binding andresidence time on the target, thus affording a higher saturation withthe drug being targeted in this invention.

In preferred embodiments, the constructs are of use for therapy ofcancer. Examples of cancers include, but are not limited to, carcinoma,lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, orlymphoid malignancies. More particular examples of such cancers arenoted below and include: squamous cell cancer (e.g., epithelial squamouscell cancer), Ewing sarcoma, Wilms tumor, astrocytomas, lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastomamultiforme, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, hepatocellular carcinoma, neuroendocrine tumors,medullary thyroid cancer, differentiated thyroid carcinoma, breastcancer, ovarian cancer, colon cancer, rectal cancer, endometrial canceror uterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulvar cancer, anal carcinoma, penile carcinoma, aswell as head-and-neck cancer. The term “cancer” includes primarymalignant cells or tumors (e.g., those whose cells have not migrated tosites in the subject's body other than the site of the originalmalignancy or tumor) and secondary malignant cells or tumors (e.g.,those arising from metastasis, the migration of malignant cells or tumorcells to secondary sites that are different from the site of theoriginal tumor).

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

DNL (Dock and Lock) Technology

The DNL method is based on the specific protein/protein interactionsbetween the regulatory (R) subunits of cAMP-dependent protein kinase(PKA) and the anchoring domain (AD) of A-kinase anchoring proteins(AKAPs) (Baillie et al., FEBS Letters. 2005; 579: 3264. Wong and Scott,Nat. Rev. Mol. Cell. Biol. 2004; 5: 959). PKA, which plays a centralrole in the signal transduction pathway triggered by the binding of cAMPto the R subunits of PKA, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymesof PKA are found with two types of R subunits (RI and RII), and eachtype has α and β isoforms (Scott, Pharmacol. Ther. 1991; 50:123). The Rsubunits have been isolated only as stable dimers and the dimerizationdomain has been shown to consist of the first 44 amino-terminal residues(Newlon et al., Nat. Struct. Biol. 1999; 6:222). Binding of cAMP to theR subunits leads to the release of active catalytic subunits for a broadspectrum of serine/threonine kinase activities, which are orientedtoward selected substrates through the compartmentalization of PKA viaits docking with AKAPs (Scott et al., J. Biol. Chem. 1990; 265:21561).

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci. USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell. Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188) and any such known AD sequence may be utilized to forma DNL complex. The amino acid sequences of the AD are quite varied amongindividual AKAPs, with the binding affinities reported for RII dimersranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA. 2003;100:4445). Interestingly, AKAPs will only bind to dimeric R subunits.For human RIIα, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

DDD of Human RIIα and AD of AKAPs as Linker Modules

We have developed a platform technology to utilize the DDD of human RIIαand the AD of AKAPs as an excellent pair of linker modules for dockingany two entities, referred to hereafter as A and B, into a noncovalentcomplex, which could be further locked into a stably tethered structurethrough the introduction of cysteine residues into both the DDD and ADat strategic positions to facilitate the formation of disulfide bonds.The general methodology of the “dock-and-lock” approach is as follows.Entity A is constructed by linking a DDD sequence to a precursor of A,resulting in a first component hereafter referred to as a. Because theDDD sequence would effect the spontaneous formation of a dimer, A wouldthus be composed of a₂. Entity B is constructed by linking an ADsequence to a precursor of B, resulting in a second component hereafterreferred to as b. The dimeric motif of DDD contained in a₂ will create adocking site for binding to the AD sequence contained in b, thusfacilitating a ready association of a₂ and b to form a binary, trimericcomplex composed of a₂b. This binding event is made irreversible with asubsequent reaction to covalently secure the two entities via disulfidebridges, which occurs very efficiently based on the principle ofeffective local concentration because the initial binding interactionsbring the reactive thiol groups placed onto both the DDD and AD intoproximity (Chimura et al., Proc. Natl. Acad. Sci. USA. 2001; 98:8480) toligate site-specifically.

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,nucleic acids, cytokines and PEG.

DDD and AD Sequence Variants

In certain embodiments, the AD and DDD sequences incorporated into theDNL complex comprise the amino acid sequences of DDD1 (SEQ ID NO:1) andAD1 (SEQ ID NO:3) below. In more preferred embodiments, the AD and DDDsequences comprise the amino acid sequences of DDD2 (SEQ ID NO:2) andAD2 (SEQ ID NO:4), which are designed to promote disulfide bondformation between the DDD and AD moieties.

DDD1 (SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 2) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 3) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 4)CGQIFYLAKQIVDNAIQQAGC

However, in alternative embodiments sequence variants AD and/or DDDmoieties may be utilized in construction of the DNL complexes. Thestructure-function relationships of the AD and DDD domains have been thesubject of investigation. (See, e.g., Burns-Hamuro et al., 2005, ProteinSci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38; Alto etal., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker et al., 2006,Biochem J 396:297-306; Stokka et al., 2006, Biochem J 400:493-99; Goldet al., 2006, Mol Cell 24:383-95; Kinderman et al., 2006, Mol Cell24:397-408, the entire text of each of which is incorporated herein byreference.)

For example, Kinderman et al. (2006) examined the crystal structure ofthe AD-DDD binding interaction and concluded that the human DDD sequencecontained a number of conserved amino acid residues that were importantin either dimer formation or AKAP binding, underlined below in SEQ IDNO:1. (See FIG. 1 of Kinderman et al., 2006, incorporated herein byreference.) The skilled artisan will realize that in designing sequencevariants of the DDD sequence, one would desirably avoid changing any ofthe underlined residues, while conservative amino acid substitutionsmight be made for residues that are less critical for dimerization andAKAP binding. Thus, a potential alternative DDD sequence of use forconstruction of DNL complexes is shown in SEQ ID NO:5, wherein “X”represents a conservative amino acid substitution. Conservative aminoacid substitutions are discussed in more detail below, but could involvefor example substitution of an aspartate residue for a glutamateresidue, or a leucine or valine residue for an isoleucine residue, etc.Such conservative amino acid substitutions are well known in the art.

Human DDD sequence from protein kinase A (SEQ ID NO: 1)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 5)XXIXIXXXLXXLLXXYXVXVLXXXXXXLVXFXVXYFXXLXXXXX

Alto et al. (2003) performed a bioinformatic analysis of the AD sequenceof various AKAP proteins to design an RII selective AD sequence calledAKAP-IS (SEQ ID NO:3), with a binding constant for DDD of 0.4 nM. TheAKAP-IS sequence was designed as a peptide antagonist of AKAP binding toPKA. Residues in the AKAP-IS sequence where substitutions tended todecrease binding to DDD are underlined in SEQ ID NO:3. Therefore, theskilled artisan will realize that variants which may function for DNLconstructs are indicated by SEQ ID NO:6, where “X” is a conservativeamino acid substitution.

AKAP-IS sequence (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA (SEQ ID NO: 6)XXXXXAXXIVXXAIXXX

Similarly, Gold (2006) utilized crystallography and peptide screening todevelop a SuperAKAP-IS sequence (SEQ ID NO:7), exhibiting a five orderof magnitude higher selectivity for the RII isoform of PKA compared withthe RI isoform. Underlined residues indicate the positions of amino acidsubstitutions, relative to the AKAP-IS sequence, that increased bindingto the DDD moiety of RIIα. In this sequence, the N-terminal Q residue isnumbered as residue number 4 and the C-terminal A residue is residuenumber 20. Residues where substitutions could be made to affect theaffinity for RIIα were residues 8, 11, 15, 16, 18, 19 and 20 (Gold etal., 2006). It is contemplated that in certain alternative embodiments,the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD moietysequence to prepare DNL constructs. Other alternative sequences thatmight be substituted for the AKAP-IS AD sequence are shown in SEQ IDNO:8-10. Substitutions relative to the AKAP-IS sequence are underlined.It is anticipated that, as with the AKAP-IS sequence shown in SEQ IDNO:3, the AD moiety may also include the additional N-terminal residuescysteine and glycine and C-terminal residues glycine and cysteine, asshown in SEQ ID NO:4.

SuperAKAP-IS (SEQ ID NO: 7) QIEYVAKQIVDYAIHQA Alternative AKAP sequences(SEQ ID NO: 8) QIEYKAKQIVDHAIHQA (SEQ ID NO: 9) QIEYHAKQIVDHAIHQA(SEQ ID NO: 10) QIEYVAKQIVDHAIHQA

Stokka et al. (2006) also developed peptide competitors of AKAP bindingto PKA, shown in SEQ ID NO:11-13. The peptide antagonists weredesignated as Ht31 (SEQ ID NO:11), RIAD (SEQ ID NO:12) and PV-38 (SEQ IDNO:13). The Ht-31 peptide exhibited a greater affinity for the RIIisoform of PKA, while the RIAD and PV-38 showed higher affinity for RI.

Ht31 (SEQ ID NO: 11) DLIFEAASRIVDAVIEQVKAAGAY RIAD (SEQ ID NO: 12)LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 13) FEELAWKIAKMIWSDVFQQC

Hundsrucker et al. (2006) developed still other peptide competitors forAKAP binding to PKA, with a binding constant as low as 0.4 nM to the DDDof the RII form of PKA. The sequences of various AKAP antagonisticpeptides is provided in Table 1 of Hundsrucker et al. (incorporatedherein by reference). Residues that were highly conserved among the ADdomains of different AKAP proteins are indicated below by underliningwith reference to the AKAP IS sequence (SEQ ID NO:3). The residues arethe same as observed by Alto et al. (2003), with the addition of theC-terminal alanine residue. (See FIG. 4 of Hundsrucker et al. (2006),incorporated herein by reference.) The sequences of peptide antagonistswith particularly high affinities for the RII DDD sequence are shown inSEQ ID NO:14-16.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA AKAP7δ-wt-pep (SEQ ID NO: 14)PEDAELVRLSKRLVENAVLKAVQQY AKAP7δ-L304T-pep (SEQ ID NO: 15)PEDAELVRTSKRLVENAVLKAVQQY AKAP7δ-L308D-pep (SEQ ID NO: 16)PEDAELVRLSKRDVENAVLKAVQQY

Carr et al. (2001) examined the degree of sequence homology betweendifferent AKAP-binding DDD sequences from human and non-human proteinsand identified residues in the DDD sequences that appeared to be themost highly conserved among different DDD moieties. These are indicatedbelow by underlining with reference to the human PKA RIIα DDD sequenceof SEQ ID NO:1. Residues that were particularly conserved are furtherindicated by italics. The residues overlap with, but are not identicalto those suggested by Kinderman et al. (2006) to be important forbinding to AKAP proteins. Thus, a potential DDD sequence is indicated inSEQ ID NO:17, wherein “X” represents a conservative amino acidsubstitution.

(SEQ ID NO: 1) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR L REAR A (SEQ ID NO: 17) XHIX IP X GL XELLQGYT X EVLR X QP X DLVEFA XX YF XXL XEX R X

The skilled artisan will realize that in general, those amino acidresidues that are highly conserved in the DDD and AD sequences fromdifferent proteins are ones that it may be preferred to remain constantin making amino acid substitutions, while residues that are less highlyconserved may be more easily varied to produce sequence variants of theAD and/or DDD sequences described herein.

In addition to sequence variants of the DDD and/or AD moieties, incertain embodiments it may be preferred to introduce sequence variationsin the antibody moiety or the linker peptide sequence joining theantibody with the AD sequence. In one illustrative example, threepossible variants of fusion protein sequences, are shown in SEQ IDNO:18-20.

(L) (SEQ ID NO: 18) QKSLSLSPGLGSGGGGSGGCG (A) (SEQ ID NO: 19)QKSLSLSPGAGSGGGGSGGCG (-) (SEQ ID NO: 20) QKSLSLSPGGSGGGGSGGCG

Production of Antibody Fragments

Methods of monoclonal antibody production are well known in the art andany such known method may be used to produce antibodies of use in theclaimed methods and compositions. Some embodiments may concern antibodyfragments. Such antibody fragments may be obtained by pepsin or papaindigestion of whole antibodies by conventional methods. For example,antibody fragments may be produced by enzymatic cleavage of antibodieswith pepsin to provide a 5S fragment denoted F(ab′)₂. This fragment maybe further cleaved using a thiol reducing agent and, optionally, ablocking group for the sulfhydryl groups resulting from cleavage ofdisulfide linkages, to produce 3.5S Fab′ monovalent fragments.Alternatively, an enzymatic cleavage using pepsin produces twomonovalent Fab fragments and an Fc fragment. Exemplary methods forproducing antibody fragments are disclosed in U.S. Pat. No. 4,036,945;U.S. Pat. No. 4,331,647; Nisonoff et al., 1960, Arch. Biochem. Biophys.,89:230; Porter, 1959, Biochem. J., 73:119; Edelman et al., 1967, METHODSIN ENZYMOLOGY, page 422 (Academic Press), and Coligan et al. (eds.),1991, CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley & Sons).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments or other enzymatic, chemical or genetic techniques also may beused, so long as the fragments bind to the antigen that is recognized bythe intact antibody. For example, Fv fragments comprise an associationof V_(H) and V_(L) chains. This association can be noncovalent, asdescribed in Inbar et al., 1972, Proc. Nat'l. Acad. Sci. USA, 69:2659.Alternatively, the variable chains may be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde. SeeSandhu, 1992, Crit. Rev. Biotech., 12:437.

Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (sFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains, connected by an oligonucleotidelinker sequence. Methods for producing sFvs are well-known in the art.See Whitlow et al., 1991, Methods: A Companion to Methods in Enzymology2:97; Bird et al., 1988, Science, 242:423; U.S. Pat. No. 4,946,778; Packet al., 1993, Bio/Technology, 11:1271, and Sandhu, 1992, Crit. Rev.Biotech., 12:437.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See Larrick et al., 1991, Methods:A Companion to Methods in Enzymology 2:106; Ritter et al. (eds.), 1995,MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION,pages 166-179 (Cambridge University Press); Birch et al., (eds.), 1995,MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, pages 137-185(Wiley-Liss, Inc.)

Chimeric and Humanized Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. Methods for constructingchimeric antibodies are well known in the art (e.g., Leung et al., 1994,Hybridoma 13:469).

A chimeric monoclonal antibody may be humanized by transferring themouse CDRs from the heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. The mouse framework regions (FR) in the chimeric monoclonalantibody are also replaced with human FR sequences. To preserve thestability and antigen specificity of the humanized monoclonal, one ormore human FR residues may be replaced by the mouse counterpartresidues. Humanized monoclonal antibodies may be used for therapeutictreatment of subjects. The affinity of humanized antibodies for a targetmay also be increased by selected modification of the CDR sequences(WO0029584A1). Techniques for production of humanized monoclonalantibodies are well known in the art. (See, e.g., Jones et al., 1986,Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen etal., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad.Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest etal., 1991, Biotechnology 9:266; Singer et al., J. Immun., 1993,150:2844.)

Other embodiments may concern non-human primate antibodies. Generaltechniques for raising therapeutically useful antibodies in baboons maybe found, for example, in Goldenberg et al., WO 91/11465 (1991), and inLosman et al., Int. J. Cancer 46: 310 (1990). In another embodiment, anantibody may be a human monoclonal antibody. Such antibodies areobtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge. In thistechnique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas. Methods for obtaining human antibodiesfrom transgenic mice are described by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994).

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50; each incorporated herein by reference). Such fully humanantibodies are expected to exhibit even fewer side effects than chimericor humanized antibodies and to function in vivo as essentiallyendogenous human antibodies. In certain embodiments, the claimed methodsand procedures may utilize human antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40, incorporated herein by reference). Human antibodies may begenerated from normal humans or from humans that exhibit a particulardisease state, such as cancer (Dantas-Barbosa et al., 2005). Theadvantage to constructing human antibodies from a diseased individual isthat the circulating antibody repertoire may be biased towardsantibodies against disease-associated antigens. In one non-limitingexample of this methodology, Dantas-Barbosa et al. (2005) constructed aphage display library of human Fab antibody fragments from osteosarcomapatients. The skilled artisan will realize that this technique isexemplary only and any known method for making and screening humanantibodies or antibody fragments by phage display may be utilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols as discussed above. A non-limiting example ofsuch a system is the XenoMouse® (e.g., Green et al., 1999, J. Immunol.Methods 231:11-23, incorporated herein by reference) from Abgenix(Fremont, Calif.). In the XenoMouse® and similar animals, the mouseantibody genes have been inactivated and replaced by functional humanantibody genes, while the remainder of the mouse immune system remainsintact.

A XenoMouse® immunized with a target antigen will produce humanantibodies by the normal immune response, which may be harvested and/orproduced by standard techniques discussed above. A variety of strains ofXenoMouse® are available, each of which is capable of producing adifferent class of antibody. Such human antibodies may be coupled toother molecules by chemical cross-linking or other known methodologies.Transgenically produced human antibodies have been shown to havetherapeutic potential, while retaining the pharmacokinetic properties ofnormal human antibodies (Green et al., 1999). The skilled artisan willrealize that the claimed compositions and methods are not limited to useof the XenoMouse® system but may utilize any transgenic animal that hasbeen genetically engineered to produce human antibodies.

Avimers

In certain embodiments, the precursors, monomers and/or complexesdescribed herein may comprise one or more avimer sequences. Avimers area class of binding proteins somewhat similar to antibodies in theiraffinities and specifities for various target molecules. They weredeveloped from human extracellular receptor domains by in vitro exonshuffling and phage display. (Silverman et al., 2005, Nat. Biotechnol.23:1493-94; Silverman et al., 2006, Nat. Biotechnol. 24:220.) Theresulting multidomain proteins may comprise multiple independent bindingdomains, that may exhibit improved affinity (in some casessub-nanomolar) and specificity compared with single-epitope bindingproteins. (Id.) In various embodiments, avimers may be attached to, forexample, DDD sequences for use in the claimed methods and compositions.Additional details concerning methods of construction and use of avimersare disclosed, for example, in U.S. Patent Application Publication Nos.20040175756, 20050048512, 20050053973, 20050089932 and 20050221384, theExamples section of each of which is incorporated herein by reference.

Phage Display

Certain embodiments of the claimed compositions and/or methods mayconcern binding peptides and/or peptide mimetics of various targetmolecules, cells or tissues. Binding peptides may be identified by anymethod known in the art, including but not limiting to the phage displaytechnique. Various methods of phage display and techniques for producingdiverse populations of peptides are well known in the art. For example,U.S. Pat. Nos. 5,223,409; 5,622,699 and 6,068,829, each of which isincorporated herein by reference, disclose methods for preparing a phagelibrary. The phage display technique involves genetically manipulatingbacteriophage so that small peptides can be expressed on their surface(Smith and Scott, 1985, Science 228:1315-1317; Smith and Scott, 1993,Meth. Enzymol. 21:228-257).

Targeting amino acid sequences selective for a given organ, tissue, celltype or target molecule may be isolated by panning (Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J.Nucl. Med. 43:159-162). In brief, a library of phage containing putativetargeting peptides is administered to an intact organism or to isolatedorgans, tissues, cell types or target molecules and samples containingbound phage are collected. Phage that bind to a target may be elutedfrom a target organ, tissue, cell type or target molecule and thenamplified by growing them in host bacteria.

Multiple rounds of panning may be performed until a population ofselective or specific binders is obtained. The amino acid sequence ofthe peptides may be determined by sequencing the DNA corresponding tothe targeting peptide insert in the phage genome. The identifiedtargeting peptide may then be produced as a synthetic peptide bystandard protein chemistry techniques (Arap et al., 1998a, Smith et al.,1985).

Aptamers

In certain embodiments, a targeting moiety of use may be an aptamer.Methods of constructing and determining the binding characteristics ofaptamers are well known in the art. For example, such techniques aredescribed in U.S. Pat. Nos. 5,582,981, 5,595,877 and 5,637,459, eachincorporated herein by reference. Methods for preparation and screeningof aptamers that bind to particular targets of interest are well known,for example U.S. Pat. No. 5,475,096 and U.S. Pat. No. 5,270,163, eachincorporated by reference.

Aptamers may be prepared by any known method, including synthetic,recombinant, and purification methods, and may be used alone or incombination with other ligands specific for the same target. In general,a minimum of approximately 3 nucleotides, preferably at least 5nucleotides, are necessary to effect specific binding. Aptamers ofsequences shorter than 10 bases may be feasible, although aptamers of10, 20, 30 or 40 nucleotides may be preferred.

Aptamers need to contain the sequence that confers binding specificity,but may be extended with flanking regions and otherwise derivatized. Inpreferred embodiments, the binding sequences of aptamers may be flankedby primer-binding sequences, facilitating the amplification of theaptamers by PCR or other amplification techniques.

Aptamers may be isolated, sequenced, and/or amplified or synthesized asconventional DNA or RNA molecules. Alternatively, aptamers of interestmay comprise modified oligomers. Any of the hydroxyl groups ordinarilypresent in aptamers may be replaced by phosphonate groups, phosphategroups, protected by a standard protecting group, or activated toprepare additional linkages to other nucleotides, or may be conjugatedto solid supports. One or more phosphodiester linkages may be replacedby alternative linking groups, such as P(O)O replaced by P(O)S, P(O)NR₂,P(O)R, P(O)OR′, CO, or CNR₂, wherein R is H or alkyl (1-20C) and R′ isalkyl (1-20C); in addition, this group may be attached to adjacentnucleotides through O or S. Not all linkages in an oligomer need to beidentical.

Conjugation Protocols

The preferred conjugation protocol is based on an alkyne-azide(preferably monosubstituted acetylene-azide), a thiol-maleimide, athiol-vinylsulfone, a thiol-bromoacetamide, or a thiol-iodoacetamidereaction that are facile at neutral or slightly acidic pH.

Suitable routes of administration of the conjugates of the preferredembodiments of the present invention include, without limitation, oral,parenteral, rectal, transmucosal, intestinal administration,intramuscular, subcutaneous, intramedullary, intrathecal, directintraventricular, intravenous, intravitreal, intraperitoneal,intranasal, or intraocular injections. The preferred routes ofadministration are parenteral. Alternatively, one may administer thecompound in a local rather than systemic manner, for example, viainjection of the compound directly into a solid tumor.

Kits

Various embodiments may concern kits containing components suitable fortreating diseased tissue in a patient. Exemplary kits may contain atleast one or more constructs as described herein. If the compositioncontaining components for administration is not formulated for deliveryvia the alimentary canal, such as by oral delivery, a device capable ofdelivering the kit components through some other route may be included.One type of device, for applications such as parenteral delivery, is asyringe that is used to inject the composition into the body of asubject. Inhalation devices may also be used. In certain embodiments, atherapeutic agent may be provided in the form of a prefilled syringe orautoinjection pen containing a sterile, liquid formulation orlyophilized preparation.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES

The invention is illustrated with examples below without limiting thescope thereof.

Example 1 Introduction of COOH Groups on Dextran

Dextran (70 kD MW) was derivatized with 5-bromohexanoic acid and 4 Msodium hydroxide at 80° C. for 3 h. The material was then acidified topH ˜4, optionally extracted with an organic solvent to remove unreactedbromohexanoic acid, and dialyzed, in a 10 kD molecular weight cut-off(MWCO) dialysis cassette, against water with 3 water changes. Theaqueous product was lyophilized. A known amount of modified dextran wastitrated against 0.1 N sodium hydroxide to estimate the number ofcarboxylic acid groups introduced. This showed that 44-to-100 COOHgroups were introduced per dextran, corresponding to 11% to 25% ofmonomeric units modified.

Example 2 Derivatization of COOH-Appended Dextran (70 kD MW)

The product of Example 1, with 44 COOH/70 kD dextran, was treated withwater soluble carbodiimide, EDC, and BOC-hydrazine, each at anequivalent corresponding to ˜50% of the COOH content. Briefly, EDCtreatment was done at an acidic pH of ˜6, and then the monoprotectedhydrazine was added and the pH was raised to 7.4. After incubation for 2to 3 h at the room temperature, the product was purified byultrafiltration using centrifugal filter with a 30 K MWCO. The recoveredproduct was determined, by titration against 0.1 N sodium hydroxide, tocontain 24 COOH/70 kD dextran. This indicated derivatization of 20 COOHmoieties as BOC hydrazide. The process was repeated with furtherderivatization using EDC and ethylene diamine such that the newintermediate now had 8 amino groups, 20 BOC hydrazide and 16 COOH perdextran. Finally, optimization was carried out for introducing ˜1reactive moiety per dextran polymer. This was done by reacting amine,BOC-hydrazide and COOH-containing dextran with varying molar equivalentsof SPDP (N-succinimidyl-3-(2-PyridylDithio)-Proprionate), and analyzingthe number of activated disulfide groups so introduced byspectrophotometrically assaying for 2 thiopyridone, at 343 nm, liberatedby reaction with dithiothreitol. This analysis showed that a 1:1 levelof activated disulfide-to-dextran substitution was obtained when using a5.3-fold molar excess of SPDP reagent.

Example 3 Sequential Derivatization of COOH-Appended Dextran (40 kD MW)to a Doxorubicin-Substituted Polymer

Dextran (40 kD) was derivatized with bromohexanoic acid and sodiumhydroxide, as in Example 1, to possess ˜60 COOH per dextran; this wasderivatized with BOC hydrazine and EDC to ˜50% level of COOH content,which was ˜30 Boc-hydrazide groups. Deprotection was carried out with 3Mhydrochloric acid, and the product was purified by ultrafiltration.Conjugation with doxorubicin was examined under conditions of pH 5 andpH 6. This showed that aqueous condition derivatization was moreefficient at pH 5, with the introduction of 20 Dox groups versus 12 Doxintroduced at pH 6. Doxorubicin content was determined from absorbanceat 496 nm and correlation with a doxorubicin standard curve.

Example 4 Sequential Derivatization of COOH-Appended Dextran (40 kD MW)to a Doxorubicin-Substituted Polymer by the ‘Click Chemistry’ Approach

Carboxyl-derivatized dextran (40 kD; ˜60 COOH) from Example 3 (0.0047mmol of dextran; 0.282 mmol w.r.t. COOH) was reacted with 2.6 mmol ofEDC and 2.1 mmol of propargylamine. The product, acetylene-addeddextran, was purified by repeated ultrafiltration-diafiltration. Theacetylene content was estimated to be 50-to-60 per 40 kd MW dextran,based on back-titration of the underivatized carboxylic acid groups.

The azide-incorporated doxorubicin hydrazone was prepared fromdoxorubicin (0.44 mmol) and 6-azidohexanoic acid hydrazide (as TFA salt;1.5 mmol) in methanol at room temperature overnight. The solvent wasevaporated off, and the excess hydrazide reagent was removed bytrituration with acetonitrile. The solid product so obtained had aretention time of 9.92 min when analyzed on a reverse phase HPLC columnusing gradient elution (100% A going to 100% B in 10 min at a flow of 3mL/min, and maintaining at 100% B for the next 5 min; A=0.3% ammoniumacetate pH 4.43; B=90% acetonitrile, 10% A; in-line absorbance detectionat 254 nm), and was 75% pure, with the remaining material mostlycomposed of unreacted doxorubicin. The product showed, in electrospraymass spectrum, peaks at m/e 696 (M−H), and m/e 732 (M+Cl), indicatingthe identity of the product. [The hydrazide reagent used herein wasprepared in 3 steps from 6-bromohexanoic acid (2 g) by first reactingwith sodium azide (1 g) in DMSO at 50° C. for 2 hr followed byextractive work up with water and ethylacetate. The ethylacetate extractwas washed sequentially with 1N HCl solution and brine and dried. Theproduct after solvent removal was re-dissolved in dichloromethane (50mL) and reacted with 2 g of EDC (10 mmol) and 1.4 g (10 mmol) ofBOC-hydrazide for 1 hour at ambient temperature. Extractive work up with1N HCl, satd. NaHCO₃, and brine, followed by drying and solvent removalfurnished the required product which was subjected to TFA-mediated BOCdeprotection using 10 mL of 1:1 TFA-CH₂Cl₂. This material was used forderivatizing doxorubicin.]

This partially-purified material was used as such for coupling toacetylene-containing dextran as follows. Acetylene-added dextran (0.1 mLof 3.35 mM) was reacted with 2 mg (1.44 μmol; 57-fold molar excess w.r.tto dextran) of doxorubicin-azide, incorporating an acid-cleavablehydrazone, 0.05 molar equiv of cupric sulfate (w.r.t. doxorubicinazide), and 0.5 molar equiv of sodium ascorbate (w.r.t. doxorubicinazide), and stirred overnight at ambient temperature. Reaction pH wasmaintained at ˜6.7. The product was purified by 3 successive UF-DF using10K MWCO centrifugal filter. The product was lyophilized to obtain 13.5mg of doxorubicin-derivatized dextran. The doxorubicin substitution wasdetermined to be 8.2 per dextran.

Scheme-2 describes the reactions.

Example 5 Preparation of SN38-20-O-glycinato-PEG-azide

0.5 g (0.9 mmol) of commercially availableO-(2-Azidoethyl)-O′—(N-diglycolyl-2-aminoethyl)heptaethyleneglycol wasactivated with 1.2 equiv. of DCC (0.186 g) and 1.2 equiv. ofN-hydroxysuccinimide (0.103 g) and catalytic amount of DMAP (0.003 g) indichloromethane (10 mL) for 30 min at ambient temperature. To this wasadded a solution of 0.42 g (0.76 mmol) of SN38-20-O-glycinate, in 10 mLdichloromethane, and DIEA (0.145 mL, 1.1 equiv.) After stirring for 30min, the product was purified by flash chromatography on silica gel(230-400 mesh) using CH₂Cl₂-MeOH gradient elution. The oily product(0.74 g, 98% yield) had HPLC retention time of 9.86 min under the HPLCconditions described in Example 4. The product was characterized byelectrospray mass spectrum. M+H at m/e 986, M+Na at m/e 1008; in thenegative ion mode, M−H at m/e 985. Calculated for C₄₅H₆₄N₇O₁₇ (M+H):986.4360; found: 986.4361.

Scheme-3 shows the synthesis.

Example 6 Preparation of N₃-PEG-Phe-Lys(MMT)-PABOCO-20-O—SN38-10-O—BOC

0.527 g (0.95 mmol) ofO-(2-Azidoethyl)-O′—(N-diglycolyl-2-aminoethyl)pheptaethyleneglycol wasactivated with 1.1 equiv. of DCC (0.182 g) and 1.2 equiv. ofN-hydroxysuccinimide (0.119 g) and catalytic amount of DMAP (0.005 g) indichloromethane (20 mL) for 30 min at ambient temperature. To thismixture was added the known Phe-Lys(MMT)-PABOH (0.58 g; 0.865 mmol),where MMT stands for monomethoxytrityl and PABOH is p-aminobenzylalcohol moieties, and DIEA (0.158 mL; 1.5 equiv). Stirred for 1 hr more,and the product was purified by flash chromatography. Yield: 84%. Massspectrum: M+H: m/e 1207. This material was coupled to 1 equivalent ofBOC—SN38-20-O-chloroformate. [The latter was prepared from BOC—SN38,triphosgene (0.4 equiv.) and DMAP (3.2 equiv) in dichloromethane, and assuch without purification.]. The title product was obtained in 60-80%yield after purification by flash chromatography. M+H: Calculated1725.7981; found: 1725.7953.

Scheme-4 shows the preparation.

Example 7 Preparation of azido-PEG-Phe-Lys(MMT)-PABOCO-20-O-glycinatoSN38

The intermediate azido-PEG-Phe-Lys(MMT)-PABOH (0.27 g; 0.22 mmol) fromExample 10 was activated with bis(nitrophenyl)carbonate (0.204 g; 3equiv.) and DIEA (1 equiv.) in dichloromethane (10 mL) for 3 days atambient temperature. Flash chromatography furnished the pure activatedproduct (yield: 69%), M+H Calc for C₇₁H₉₀N₉O₁₉: 1372.6347; found:1372.6347. Activated carbonate product (0.08 g; 0.058 mmol) was coupledto SN38-20-O-glycinate (0.028 g; 0.058 mmol) in DMF (1 mL) and DIEA(0.025 mL; 2.5 equiv.). After 4 h of stirring, solvent was removed andthe crude product was purified by flash chromatography. Yield: 0.052 g(54%). M+H Calc for C₈₉H₁₀₈N₁₁O₂₂: 1682.7665; found: 1682.7682.

Scheme-5 describes the reactions.

Example 8 Derivatization of Succinimidyl 4-Maleimidomethyl-CyclohexaneCarboxylate (SMCC) with N—BOC-2,2′-(Ethylenedioxy)Diethylamine, Followedby BOC-Deprotection

SMCC (0.334 g), monoprotected diamine reagent (0.248 g) and DIEA (0.17mL) were dissolved in dichloromethane (20 mL), stirred at ambienttemperature for 20 min. The product was purified by flashchromatography, and further reacted with TFA (2 mL) and anisole (0.5 mL)for 2 hours, and the final product was isolated after removal of TFA andanisole. The corresponding hydrochloride salt was prepared by dissolvingin HCl and evaporating off HCl. Mass spectrum: M+H m/e 368. The processschematically shown in Scheme-6.

Example 9 Derivatization of Acetylene-Containing Dextran of Example-4with the Product of Example 8

To an aqueous solution of acetylene-dextran (40 KD MW; 0.425 g) in 10 mLof water, added product of example 8 (0.085 g; 20 equiv. w.r.t dextran)and EDC (0.0406 g; 20 equiv.), stirred for 1 hour. Purified byultrafiltration-diafiltration using 10 kd MW CO filter. Anthrone assayfor dextran showed the dextran concentration to be 28.6 mg/mL. RevereseEllman's assay using excess of 2-mercaptoethanol and determining theexcess unsused 2-ME by Ellman's assay gave a value of 5.4 maleimidessubstituted on to dextran. Scheme-7 depicts the reactions.

Example 10 Click Chemistry Coupling ofDextran-Acetylene₍₅₀₋₆₀₎-maleimide_((5,5)) withSN38-20-O-Glycinato-PEG-Azide Products of Example 5 or Example 6 orExample 7

10 mL of 28.6 mg/mL solution of the dextran derivative of Example 9 wasreacted with 0.42 M DMSO solution of the SN38 derivative specified inExample 5 (70 equiv.) in the presence of a catalytic amount of cupricsulfate and sodium ascorbate in a 10-fold excess over copper sulfate.DMSO concentration was 20% v/v. The somewhat cloudy solution was stirredfor 4 hr. The product was purified by ultrafiltration/diafiltration,using 0.2 M aqueous EDTA, followed by gel filtration. The product wascharacterized by anthrone assay (10.74 mg/mL), and SN38 concentrationwas determined by absorbance at 366 nm and correlation with a standardcurve. SN38 molar substitution was calculated to be 36.6. Free unremovedSN38 level was estimated to be 5% by HPLC. The product of reaction usingazide-SN38 of Example 5 is illustrated below in Scheme-8.

In a similar fashion, the dextran derivative of Example 9 is reactedwith the azido-SN38 derivative of Examples 6 or 7 to obtain thecorresponding dextran conjugates. In these cases, the BOC and MMTprotecting groups are subsequently removed by treatment with 2 Nhydrochloric acid or by a short-duration treatment (<5 min) withtrifluoroacetic acid. Alternatively, the protecting groups are removedfirst, followed by click chemistry coupling to the dextran derivative ofExample 9.

Example 11 Coupling of any Dextran Derivative of Example 10 with aThiol-Containing Material Incorporating a Recognition Moiety

The reaction is done by coupling a maleimide-appended dextran of Example10 with 5.4 equivalent of the recognition moiety-incorporated,thiol-containing peptide in 75 mM sodium acetate-1 mM EDTA, pH 6.5, for1 hr. For pretargeting, prototypical peptide in this regard isAc-Cys-(AA)_(n)-Lys(HSG)-NH₂, wherein AA is an amino acid, and n is aninteger from 1-20, preferably 1-3. One of the amino acids represented by‘AA’ can be lysine with HSG substituted on the lysine side chain aminogroup, thereby making the peptide a bis-HSG-containing peptide. Thesubstitution of the N-terminal cysteine can be a chelator such asbenzyl-DTPA, instead of acyl, for determining by metal-binding assaysthe number of peptides attached to the polymer. For DNL coupling, thepeptide is cysteine-containing anchoring domain (‘AD’) peptide, such asillustrated in paragraph 0051. The other recognition moieties describedin paragraph 0014 are also useful in this reaction after suitable priorderivatization of the same to possess a thiol group. The product ispurified by ultrafiltration-diafiltration, followed by centrifugedsize-exclusion column chromatography using non-EDTA buffer. Using anHSG-incorporated peptide, which further contains a metal chelator,metal-binding assay gives a chelator content of 2.5 per dextran. Thissuggests that at least 2.5 mole per mole of dextran is accessible forreaction with thiol-containing material. A test labeling with In-111acetate is done, and the material is purified by size-exclusionchromatography. HPLC analysis of the radiolabeled material as well asthat of the material complexed with anti-HSG antibody (murine 679) showscomplete complexation, as revealed by the shift of the SE HPLC peak dueto In-111-dextran to a peak due to the higher MW of the dextran: 679antibody complex. The unlabeled material is also shown to be complexedwith murine 679 antibody, as the broad size-exclusion HPLC peak due todextran derivative is shifted to a relatively sharper and faster elutingpeak, indicating complexation with murine 679 antibody. The conjugationto HSG-containing peptide is given in Scheme-9.

Example 12 Derivatizations of Polyglutamic Acid

Poly-L-glutamic acid (PG) is reacted with EDC and propargylamine. Theproduct, acetylene-added PG is then purified by repeatedultrafiltration-diafiltration. The acetylene content is estimated byback-titration of the underivatized carboxylic acid groups. Theacetylene-appended PG is sequentially derivatized with themaleimide-containing amino compound of Example 8 by EDC-mediatedcoupling to COOH groups of PG, followed by acetylene-azide couplingusing azide-derivatized doxorubicin of Examples 3 or 4, orazide-derivatized SN-38 of Examples 5, 6, or 7. The respective productis purified by ultrafiltration-diafiltration. When the azide-drug is ofExample 6 or 7, a further deprotection of BOC and MMT groups is alsocarried out with hydrochloric acid or trifluoroacetic acid, as describedin paragraph 0084. Finally, the material is derivatized with athiol-containing recognition-moiety, as described in Example 11. PGswith molecular weights in the ranges of 750-5000, 3000-15,000,15,000-50,000, and 50,000-100,000 are used in this context.

What is claimed is:
 1. A method of preparing a modified dendrimer comprising: (a) obtaining a dendrimer molecule comprising surface carboxylic acid groups; (b) reacting one or more of the carboxylic acid groups with a carbodiimide and (i) an amine-containing acetylene molecule, or (ii) an amine-containing azide molecule to form an acetylene-derivatized or azide-derivatized dendrimer molecule; (c) reacting other carboxylic acid groups of the acetylene-derivatized or azide-derivatized dendrimer molecule with a carbodiimide and a maleimido amine to form a maleimide-appended, acetylene- or azide-derivatized dendrimer molecule; (d) reacting (i) the maleimide-appended, acetylene-derivatized dendrimer molecule with an azide-derivatized drug or (ii) the maleimide-appended, azide-derivatized dendrimer molecule with an acetylene-derivatized drug to form a maleimide-appended, drug modified dendrimer molecule; and (e) coupling the maleimide-appended, drug modified dendrimer molecule with a thiol-containing compound comprising a recognition moiety to form a recognition moiety-appended, drug-modified dendrimer molecule.
 2. The method of claim 1, wherein the drug is attached to the dendrimer by a click chemistry reaction.
 3. The method of claim 1, wherein the dendrimer is attached to two or more different drugs.
 4. The method of claim 3, wherein the two different drugs are SN-38 and doxorubicin.
 5. The method of claim 1, wherein the drug is attached to the dendrimer with a cleavable linker.
 6. The method of claim 1, wherein the recognition moiety is selected from the group consisting of (i) a peptide comprising one or more hapten moieties; (ii) folic acid; (iii) a targeting peptide; (iv) biotin; (v) an oligonucleotide; (vi) an anchoring domain peptide; (vii) an antibody; and (viii) an antibody fragment.
 7. The method of claim 6, wherein the hapten is HSG or In-DTPA.
 8. The method of claim 6, wherein the targeting peptide is somatostatin or VIP.
 9. The method of claim 1, wherein the dendrimer is attached to 1 to 10 recognition moieties.
 10. The method of claim 9, wherein the dendrimer is attached to 1 to 2 recognition moieties.
 11. The method of claim 9, wherein the dendrimer is attached to 1 recognition moiety.
 12. The method of claim 1, wherein the dendrimer is derivatized with an amine-containing acetylene molecule in step (b), and step (d) involves an azide-derivatized drug.
 13. The method of claim 1, wherein the dendrimer is derivatized with amine-containing azide molecule in step (b), and step (d) involves an acetylene-derivatized drug.
 14. The method of claim 1, wherein the drug is selected from the group consisting of chemotherapeutic drugs, vinca alkaloids, anthracyclines, epipodophyllotoxins, taxanes, antimetabolites, alkylating agents, antibiotics, Cox-2 inhibitors, antimitotic agents, antiangiogenic agents, pro-apoptotic agents, doxorubicin, methotrexate, paclitaxel, camptothecins, nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purine analogs, platinum coordination complexes, hormones, toxins, ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin and Pseudomonas endotoxin.
 15. The method of claim 1, wherein the drug is selected from the group consisting of 5-fluorouracil, aplidin, azaribine, anastrozole, anthracyclines, bendamustine, bleomycin, bortezomib, bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, estramustine, epipodophyllotoxin, estrogen receptor binding agents, etoposide (VP16), etoposide glucuronide, etoposide phosphate, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl-protein transferase inhibitors, gemcitabine, hydroxyurea, idarubicin, ifosfamide, L-asparaginase, lenalidomide, leucovorin, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine, nitrosourea, plicamycin, procarbazine, paclitaxel, pentostatin, PSI-341, raloxifene, semustine, streptozocin, tamoxifen, taxol, temazolomide, transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vinorelbine, vinblastine, vincristine and vinca alkaloids.
 16. The method of claim 1, wherein the drug is doxorubicin or SN-38.
 17. The method of claim 5, wherein the cleavable linker comprises a hydrazone, a cathepsin-B-cleavable peptide, a disulfide or an ester bond.
 18. The method of claim 1, wherein the drug is selected from the group consisting of SN-38-20-O-glycinato-PEG-azide, N3-PEG-Phe-Lys(monomethoxytrityl)-PABOCO-20-O—SN-38-10-O—BOC and azido-PEG-Phe-Lys(monomethoxytrityl)-PABOCO-20-O-glycinato-SN-38, wherein the method further comprises removing the BOC and monomethoxytrityl protecting groups from the recognition moiety-appended, drug-modified dendrimer molecule.
 19. The method of claim 6, wherein the antibody is selected from the group consisting of R1, RS7, LL1, LL2, RFB4, A20, A19, IMMU31, RS7, PAM4, KC4, MN-3, MN-14, MN-15, Mu-9, Immu 31, CC49, Tn, J591, G250 and L243.
 20. The method of claim 6, wherein the antibody or antibody fragment binds to an antigen selected from the group consisting of EGP-1, EGP-2, CEA (CEACAM5), CEACAM6, CSAp, carbonic anhydrase IX, HER-2/neu, BrE3, CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD74, CD80, CD126, CXCR4, alpha-fetoprotein (AFP), B7, VEGFR, ED-B, EGF receptor (ErbB1), ErbB2, ErbB3, HM1.24, HCG, placental growth factor (PlGF), MUC1, MUC2, MUC3, MUC4, MUC5, MUC16, PSMA, gangliosides, HLA-DR, Ia, A3, A33, Ep-CAM, KS-1, Le(y), 5100, PSA (prostate-specific antigen), tenascin, folate receptor, Thomas-Friedenreich antigens, tumor necrosis antigens, tumor angiogenesis antigens, Ga 733, IL-2, IL-6, T101, TAC, MAGE, insulin-like growth factor (IGF), MIF, CD66a, CD66b, CD66c and CD66d. 